Propofol suppresses microglial phagocytosis through the downregulation of MFG-E8.

BACKGROUND: Microglia are highly motile phagocytic cells in the healthy brain with surveillance and clearance functions. Although microglia have been shown to engulf cellular debris following brain insult, less is known about their phagocytic function in the absence of injury. Propofol can inhibit microglial activity, including phagocytosis. Milk fat globule epidermal growth factor 8 (MFG-E8), as a regulator of microglia, plays an essential role in the phagocytic process. However, whether MFG-E8 affects the alteration of phagocytosis by propofol remains unknown. METHODS: Microglial BV2 cells were treated with propofol, with or without MFG-E8. Phagocytosis of latex beads was evaluated by flow cytometry and immunofluorescence. MFG-E8, p-AMPK, AMPK, p-Src, and Src levels were assessed by western blot analysis. Compound C (AMPK inhibitor) and dasatinib (Src inhibitor) were applied to determine the roles of AMPK and Src in microglial phagocytosis under propofol treatment. RESULTS: The phagocytic ability of microglia was significantly decreased after propofol treatment for 4 h (P < 0.05). MFG-E8 production was inhibited by propofol in a concentration- and time-dependent manner (P < 0.05). Preadministration of MFG-E8 dose-dependently (from 10 to 100 ng/ml) reversed the suppression of phagocytosis by propofol (P < 0.05). Furthermore, the decline in p-AMPK and p-Src levels induced by propofol intervention was reversed by MFG-E8 activation (P < 0.05). Administration of compound C (AMPK inhibitor) and dasatinib (Src inhibitor) to microglia blocked the trend of enhanced phagocytosis induced by MFG-E8 (P < 0.05). CONCLUSIONS: These findings reveal the intermediate role of MFG-E8 between propofol and microglial phagocytic activity. Moreover, MFG-E8 may reverse the suppression of phagocytosis induced by propofol through the regulation of the AMPK and Src signaling pathways.

MFG-E8 alleviates oxygen-glucose deprivation-induced neuronal cell apoptosis by STAT3 regulating the selective polarization of microglia.

Background: Ischemic stroke is a complex pathological process, involving inflammatory reaction, energy metabolism disorder, free radical injury, cell apoptosis and other aspects. Accumulating evidences have revealed that MFG-E8 had a protective effect on multiple organ injuries. However, the comprehensive function and mechanism of MFG-E8 in ischemic brain remain largely unclear.Methods: BV-2 cells were treated with recombinant murine MFG-E8 (rmMFG-E8) or/and Colivelin TFA after exposing for 4 h with oxygen glucose deprivation (OGD). Cell viability and apoptosis were assessed by MTT assay and Flow cytometry. RT-qPCR and Western blot assays were applied to examine the expression levels of MFG-E8, apoptosis-related proteins and M1/M2 polarization markers.Results: Our results demonstrated that OGD significantly inhibited microglial viability and facilitated apoptosis. In addition, we found that OGD downregulated MFG-E8 expression, and MFG-E8 inhibited OGD-induced microglial apoptosis and promoted microglial M2 polarization. In terms of mechanism, we proved that MFG-E8 regulated OGD-induced microglial M1/M2 polarization by inhibiting p-STAT3 and SOCS3 expressions, which was reversed by STAT3 activator (Colivelin TFA). Finally, we verified MFG-E8 alleviated OGD-induced neuronal cell apoptosis by M2 polarization of BV-2 cells.Conclusions: We demonstrated that MFG-E8 reduced neuronal cell apoptosis by enhancing activation of microglia via STAT3 signaling. Therefore, we suggested that MFG-E8 might provide a novel mechanism for ischemic stroke.

The significance of tumor cells-derived MFG-E8 in tumor growth of angiosarcoma.

BACKGROUND: Previous studies have indicated that MFG-E8 enhances tumor cell survival, invasion and angiogenesis. However, the role of MFG-E8 in angiosarcoma (AS) has not been clarified. OBJECTIVE: Objective was to elucidate the mechanism of the regulation by MFG-E8 in AS and the association between MFG-E8 and clinicopathological features of AS. METHODS: The effects of the depletion of MFG-E8 by siRNA on tube formation, migration and proliferation in murine AS cells were examined. The effect of administration of anti-MFG-E8 antibody (Ab) on tumor growth of AS in mice was examined. The associations of MFG-E8 expression and clinicopathological features of human AS were assessed. RESULTS: The expressions of MFG-E8 in murine and human AS cells were significantly higher than those in melanoma cells, macrophages and endothelial cells. Depletion of MFG-E8 in murine AS cells by siRNA significantly inhibited the formation of capillary-like structures and migration, but not proliferation. Administration of anti-MFG-E8 Ab significantly inhibited tumor growth and decreased the number of tumor-associated macrophages (TAMs) in AS tumors. Tumor size and the number of TAMs in human AS with high expression of MFG-E8 were significantly increased compared to those of AS with low expression of MFG-E8. Progression-free survival and overall survival time of the patients of AS with high expression of MFG-E8 were significantly shorter than those of AS with low expression of MFG-E8. CONCLUSIONS: AS-derived MFG-E8 might enhance tumor growth via angiogenesis and the induction of TAMs in autocrine/paracrine manner, and administration of anti-MFG-E8 Ab could be a therapeutic potential for AS.

Potential cow milk xanthine oxidase inhibitory and antioxidant activity of selected phenolic acid derivatives.

Xanthine oxidase (XO) found in all mammals and excess activity leads to urolithiasis. The cow milk XO was purified to 305-fold with a specific activity of 8.76 EU/mg and overall yield of 87% by using DEAE-Sepharose chromatography. The phenolics showed potent XO inhibitory effect with Ki , P1 (0.412), P2 (0.632), P3 (0.585), P4 (0.886), P5 (1.633), P6 (0.503), P7 (2.882), P8 (3.761), P9 (4.487), and P10 (5.841) µM. The phenolics P9 and P10 exhibited uncompetitive inhibition; the phenolics P1, P2, P3, P4, and P6 showed competitive inhibition, and other phenolic acids showed noncompetitive inhibition. The studied phenolic compounds showed potent antioxidant activity and expressed as EC50 , ranged from, DPPH (4.2-25.8 µg mL-1 ), ABTS (10.2-42.5 mmol TE 100 g-1 ), and FRAP (6.3-36.8 mol Fe (II) 100 g-1 ). The results obtained from this study might be utilized for design of XO inhibitors and as antigout agent.

Antioxidant, anti-inflammatory, and enzyme inhibitory activity of natural plant flavonoids and their synthesized derivatives.

The synthesized flavonoid derivatives were examined for their antioxidant, anti-inflammatory, xanthine oxidase (XO), urease inhibitory activity, and cytotoxicity. Except few, all the flavonoids under this study showed significant antioxidant activity (45.6%-85.5%, 32.6%-70.6%, and 24.9%-65.5% inhibition by DPPH, ferric reducing/antioxidant power, and oxygen radical absorption capacity assays) with promising TNF-α inhibitory activity (42%-73% at 10 µM) and IL-6 inhibitory activity (54%-81% at 10 µM) compared with that of control dexamethasone. The flavonoids luteolin, apigenin, diosmetin, chrysin, O3Ꞌ , O7 -dihexyl diosmetin, O4Ꞌ , O7 -dihexyl apigenin, and O7 -hexyl chrysin, showed an inhibition with IC50 values (4.5-8.1 µg/mL), more than allopurinol (8.5 µg/mL) at 5 µM against XO and showing more than 50% inhibition at a final concentration (5 mM) with an IC50 value of ranging from 4.8 to 7.2 (µg/mL) in comparison with the positive control thiourea (5.8 µg/mL) for urease inhibition. Thus, the flavonoid derivatives may be considered as potential antioxidant and antigout agents.

Potent Inhibitory Effects of Some Phenolic Acids on Lactoperoxidase.

Lactoperoxidase (LPO) plays a key role in immune response against pathogens. In this study, we examined the effects of some phenolic acids on LPO. For this purpose, bovine milk LPO was purified 380.85-fold with a specific activity of 26.66 EU/mg and overall yield of 73.33% by using Amberlite CG-50 H+ resin and CNBr-activated Sepharose-4B-l-tyrosine-sulfanilamide affinity chromatography. After purification, the in vitro effects of phenolic acids (tannic acid, 3,4-dihydroxybenzoic acid, 3,5- dihydroxybenzoic acid, chlorogenic acid, sinapic acid, 4-hydroxybenzoic acid, vanillic acid, salicylic acid, and 3-hydroxybenzoic acid) were investigated on LPO. These phenolic acids showed potent inhibitory effect on LPO. Ki values for these phenolic acids were found as 0.0129 nM, 0.132 µM, 0.225 µM, 0.286 µM, 0.333 µM, 2.33 µM, 10.82 µM, 0.076 mM, and 0.405 mM, respectively. Sinapic acid and 4-hydroxybenzoic acid exhibited noncompetitive inhibition; 3,4-dihydroxybenzoic acid showed uncompetitive inhibition, and other phenolic acids showed competitive inhibition.

Down-regulation of MFG-E8 by RNA interference combined with doxorubicin triggers melanoma destruction.

The pathogenic mechanism of malignant melanoma involves the dynamic interplay of transformed cell and normal host cell, but cancer treatments always target each partition separately. In the tumor microenvironment, milk fat globule epidermal growth factor-8 (MFG-E8) is a secreted glycoprotein highly expressed in the vertical growth phase of melanoma, leading to tumor progression through coordinated αvß3 and αvß5 integrin signaling in tumor cells and host cells. Doxorubicin (Dox) is one of the most widely used antitumor drugs against a lot of solid tumors, including melanoma. In this work, Dox was used to combine with down-regulation of MFG-E8 by RNA interference (RNAi) in order to determine the synergistic effect of the antitumor activity in vivo. And the possible mechanisms were investigated. Results showed that combination group (MFG-E8 RNAi plus Dox) could inhibit the growth of melanoma more effectively than monotherapy or control groups. We found that the combination treatment induced more tumor cell apoptosis and inhibited more neovascularization than other groups. Moreover, this combination treatment attenuated CD4(+) CD25(+) Foxp3(+) Treg cells in tumor-infiltrating lymphocytes compared with other groups. Our findings suggested that MFG-E8 down-regulation enhanced the antitumor function of chemotherapy through coordinated cell apoptosis and immune-mediated mechanisms, which might be a feasible way for cancer therapy.

Role for the endometrial epithelial protein MFG-E8 and its receptor integrin αvß3 in human implantation: results of an in vitro trophoblast attachment study using established human cell lines.

OBJECTIVE: To investigate the role of MFG-E8 and its receptor integrin αvß3 in the attachment of trophoblast cells to the endometrial epithelium. DESIGN: Experimental in vitro study. SETTING: Academic center. PATIENT(S): None. INTERVENTION(S): By using a well-differentiated endometrial adenocarcinoma cell line (Ishikawa cells) and choriocarcinoma human trophoblast cells (Jar cells), an in vitro assay mimicking human implantation was established. To investigate the impact of blocking MFG-E8 and integrin αvß3, we pretreated the cell lines with antibodies against those proteins at different concentrations before the attachment assay. MAIN OUTCOME MEASURE(S): Attachment rate of Jar spheroids to the epithelial cell monolayer. RESULT(S): Pretreatment of Ishikawa cells with anti-MFG-E8 antibody caused a dose-dependent and significant inhibition of attachment. On the other hand, pretreatment of Jar spheroids did not result in a significant effect on the attachment rate. Pretreatment of Ishikawa cells as well as Jar spheroids with anti-integrin αvß3 antibodies resulted in a dose-dependent, significant inhibition of attachment. CONCLUSION(S): This study showed that blocking MFG-E8 and its receptor integrin αvß3 in Ishikawa cells diminishes Jar spheroid attachment. Moreover, blocking integrin αvß3 in the trophoblastic cells also diminished their attachment to the Ishikawa monolayer.

Essential role of MFG-E8 for phagocytic properties of microglial cells.

Milk fat globule factor-E8 (MFG-E8) has been regarded as a key factor involved in the phagocytosis of apoptotic cells. We induced a lentivirus into the microglial cells for the augmentation or abrogation of MFG-E8 expression in mouse microglial cells, and investigated phagocytosis of phosphatidylserine tagged human red blood cells (hRBCs) in co-cultures. Increased MFG-E8 levels were associated with a significant increase in phagocytic activity compared to the controls. Conversely, phagocytosis dramitically decreased due to the abrogation of MFG-E8. In addition, the expression of the inflammatory cytokines, TNF-α and IL-1ß, also increased or decreased in the microglial cells with the augmentation or abrogation of MFG-E8, respectively. Our findings indicate that the enhanced expression of MFG-E8 could increase phagocytosis of apoptotic cells; conversely, the rate of phagocytosis and the expression of inflammatory cytokines decreased when MFG-E8 expression was knocked down. Our results confirm that MFG-E8 plays an important role in phagocytosis, and possibly serves as an essential signal molecule for microglial cells.

Hepatoprotection by chemical constituents of the marine brown alga Spatoglossum variabile: a relation to free radical scavenging potential.

CONTEXT: In the course of searching hepatoprotective agents from natural sources, the protective effect of chemical constituents of the marine brown alga Spatoglossum variabile Figaro et DE Notar (Dictyoaceae) against CCl4-induced liver damage in Wistar rats was investigated. The compounds were first investigated for in vitro radical scavenging potential and were also tested for ß-glucuronidase inhibition to further explore the relationship between hepatoprotection and antiradical potential. METHODS: The compounds cinnamic acid esters 1 and 2 and aurone derivatives 3 and 4 were first investigated for in vitro radical scavenging potential against 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH), and superoxide anion radicals. In vivo hepatoprotective studies were performed in seven groups (n = 6) of Wistar rats. The test groups were pretreated with compounds (10 mg/kg body weight, po) orally for 30 min before the intraperitoneal administration of a dose of 20% CCl4 diluted with dietary cooking oil. Moreover, compounds were also tested for ß-glucuronidase inhibition to explore the relationship between hepatoprotection and radical scavenging potential. RESULTS: The test compounds 1-4 were found to exhibit antiradical activity against 1,1-diphenyl-2-picrylhydrazyl radicals with IC50 values ranging between 54 and 138 µM, whereas aurone derivatives 3 and 4 additionally exhibited superoxide anion scavenging effects with IC50 values of 95 and 87 µM, respectively. In addition, these compounds were found to be weak inhibitors of xanthine oxidase (IC50 ≥1000 µM). In animal model, pretreatment with compounds 2-4 significantly blocked the CCl4-induced increase in the levels of the serum biochemical markers. CONCLUSION: It appears that the hepatoprotection afforded by these compounds was mainly due to their radical scavenging activity that protected the cells from the free radicals generated by CCl4-induced hepatotoxicity.

Post-weaning increases in the milk-fat globule EGF-factor VIII on fat globules in mouse milk and in the uptake of the fat globules by HC11 mammary epithelial cells.

Milk fat globules (MFGs) secreted by lactating mammary gland are unique lipid surrounded by a phospholipid bi-layer. We report here post-weaning changes in MFG EGF factor VIII (MFG-E8) and annexin V-accessible phosphatidyl-l-serine on the surface of MFGs. The MFG content in milk markedly decreased to about one-half within 2 days after forced weaning, despite a slight increase in milk protein content. Immunofluorescence-staining of MFGs using anti-MFG-E8 and annexin V indicated that MFG-E8 was present on some, but not all, MFGs before weaning, whereas most of MFGs were MFG-E8-positive and annexin V-negative after weaning. Free MFG-E8 with binding activity to phosphatidyl-l-serine was present abundantly in the post-weaning milk, and indeed exhibited binding to MFGs in pre-weaning milk. MFGs were taken up by HC11 mouse mammary epithelial cells in vitro, and those from post-weaning milk were remarkable for such cellular uptake. Moreover, the uptake of MFGs by the cells was inhibited by an anti-MFG-E8 antibody. Taken together, these findings suggest that MFG-E8 plays a critical role in regulation of MFG dynamics after weaning or during the suckling interval through the control of MFG-epithelial cell interaction in lactating mammary glands.

Potentiation of platelet-derived growth factor receptor-ß signaling mediated by integrin-associated MFG-E8.

OBJECTIVE: Pericytes/pericyte precursors produce milk fat globule-associated protein with epidermal growth factor and factor VIII-like domains (MFG-E8) in vivo, and this α(v) integrin ligand enhances angiogenesis in tumors and in oxygen-induced retinopathy in mice. Inhibition of MFG-E8 production or function attenuates platelet-derived growth factor-BB (PDGF-BB)-induced migration of pericyte/pericyte precursor-like 10T1/2 cells in vitro. Herein, we describe mechanisms by which MFG-E8 modulates PDGF-BB:PDGF receptor ß (PDGFRß) signaling in 10T1/2 cells. METHODS AND RESULTS: Small interfering RNA depletion of MFG-E8 from 10T1/2 cells or antibody inhibition of MFG-E8 action enhanced PDGF-BB-dependent degradation of PDGFRß and attenuated signaling. Coimmunoprecipitation revealed transient association of MFG-E8 with PDGFRß in PDGF-BB-treated 10T1/2 cells and reduced PDGFRß-focal adhesion kinase association in MFG-E8-depleted cells. Confocal microscopy demonstrated that MFG-E8 binding to 10T1/2 cells was RGD motif and α(v) dependent but PDGF-BB treatment independent, whereas colocalization of MFG-E8 with PDGFRß was enhanced by PDGF-BB. Ubiquitination of PDGFRß was also increased in MFG-E8 small interfering RNA-transfected cells. CONCLUSION: Integrin α(v)-bound MFG-E8 associates with PDGFRß and focal adhesion kinase after PDGF-BB treatment, results in cell surface retention of PDGFRß, delays receptor degradation, potentiates downstream signaling, and enhances migration of 10T1/2 cells. MFG-E8 may promote angiogenesis, in part, via cell autonomous actions on pericytes or pericyte precursors that result in enhanced PDGF-BB:PDGFRß signaling mediated via integrin-growth factor receptor cross-talk.

Grape seed procyanidin B2 inhibits advanced glycation end product-induced endothelial cell apoptosis through regulating GSK3ß phosphorylation.

To investigate the effects of GSPB2 (grape seed procyanidin B2) on the apoptosis of HUVECs (human umbilical endothelial cells) induced by AGEs (advanced glycation end products), HUVECs were treated with AGEs (200 µg/ml) in the presence or absence of GSPB2 (2.5, 5.0 and 10.0 µmol/l). Our findings showed that (i) AGEs induced HUVEC apoptosis and up-regulated the expression of caspase-3 activation and lactadherin and reduced the phosphorylation of GSK3ß (glycogen synthase kinase 3ß) at baseline. (ii) Treatment of HUVEC with GSPB2 significantly inhibited the cell apoptosis and the expression of caspase-3 activation and lactadherin induced by AGEs. Moreover, GSPB2 inhibited intracellular reactive oxygen species in a dose-dependent manner in AGEs-treated cells as determined by flow cytometry. (iii) GSPB2 increased the phosphorylation of GSK3ß of HUVEC in response to AGEs. These findings suggest that the signalling pathway involving phosphorylation of GSK3ß and lactadherin might play a key role in the endothelial apoptosis. GSPB2 therapy could become an effective approach to battling AGEs-induced endothelial apoptosis.

Engulfment of cerebral apoptotic bodies controls the course of prion disease in a mouse strain-dependent manner.

Progressive accumulation of PrP(Sc), a hallmark of prion diseases, occurs when conversion of PrP(C) into PrP(Sc) is faster than PrP(Sc) clearance. Engulfment of apoptotic bodies by phagocytes is mediated by Mfge8 (milk fat globule epidermal growth factor 8). In this study, we show that brain Mfge8 is primarily produced by astrocytes. Mfge8 ablation induced accelerated prion disease and reduced clearance of cerebellar apoptotic bodies in vivo, as well as excessive PrP(Sc) accumulation and increased prion titers in prion-infected C57BL/6 × 129Sv mice and organotypic cerebellar slices derived therefrom. These phenotypes correlated with the presence of 129Sv genomic markers in hybrid mice and were not observed in inbred C57BL/6 Mfge8(-/-) mice, suggesting the existence of additional strain-specific genetic modifiers. Because Mfge8 receptors are expressed by microglia and depletion of microglia increases PrP(Sc) accumulation in organotypic cerebellar slices, we conclude that engulfment of apoptotic bodies by microglia may be an important pathway of prion clearance controlled by astrocyte-borne Mfge8.

Interactions of melatonin and serotonin with lactoperoxidase enzyme.

Melatonin is the chief secretory product of the pineal gland and is synthesized enzymatically from serotonin. These indoleamine derivatives play an important role in the prevention of oxidative damage. Lactoperoxidase (LPO; EC 1.11.1.7) was purified from bovine milk with three purification steps: Amberlite CG-50 resin, CM-Sephadex C-50 ion-exchange, and Sephadex G-100 gel filtration chromatography, respectively. LPO was purified with a yield of 21.6%, a specific activity of 34.0 EU/mg protein, and 14.7-fold purification. To determine the enzyme purity, SDS-PAGE was performed and a single band was observed. The R(z) (A(412)/A(280)) value for LPO was 0.9. The effect of melatonin and serotonin on lactoperoxidase was determined using ABTS as chromogenic substrate. The half-maximal inhibitory concentration (IC(50)) values for melatonin and serotonin were found to be 1.46 and 1.29 µM, respectively. Also, the inhibition constants (K(i)) for melatonin and serotonin were 0.82 ± 0.28 and 0.26 ± 0.04 µM, respectively. Both melatonin and serotonin were found to be competitive inhibitors.

Immature dendritic cell-derived exosomes rescue septic animals via milk fat globule epidermal growth factor-factor VIII [corrected].

Sepsis, a highly lethal systemic inflammatory syndrome, is associated with increases of proinflammatory cytokines (e.g., TNF-alpha, HMGB1) and the accumulation of apoptotic cells that have the potential to be detrimental. Depending on the timing and tissue, prevention of apoptosis in sepsis is beneficial; however, thwarting the development of secondary necrosis through the active removal of apoptotic cells by phagocytosis may offer a novel anti-sepsis therapy. Immature dendritic cells (IDCs) release exosomes that contain milk fat globule EGF factor VIII (MFGE8), a protein required to opsonize apoptotic cells for phagocytosis. In an experimental sepsis model using cecal ligation and puncture, we found that MFGE8 levels decreased in the spleen and blood, which was associated with impaired apoptotic cell clearance. Administration of IDC-derived exosomes promoted phagocytosis of apoptotic cells and significantly reduced mortality. Treatment with recombinant MFGE8 was equally protective, whereas MFGE8-deficient mice suffered from increased mortality. IDC exosomes also attenuated the release of proinflammatory cytokines in septic rats. Liberation of HMGB1, a nuclear protein that contributes to inflammation upon release from unengulfed apoptotic cells, was prevented by MFGE8-mediated phagocytosis in vitro. We conclude that IDC-derived exosomes attenuate the acute systemic inflammatory response in sepsis by enhancing apoptotic cell clearance via MFGE8.

Milk fat globule epidermal growth factor-8 blockade triggers tumor destruction through coordinated cell-autonomous and immune-mediated mechanisms.

Carcinogenesis reflects the dynamic interplay of transformed cells and normal host elements, but cancer treatments typically target each compartment separately. Within the tumor microenvironment, the secreted protein milk fat globule epidermal growth factor-8 (MFG-E8) stimulates disease progression through coordinated alpha(v)beta(3) integrin signaling in tumor and host cells. MFG-E8 enhances tumor cell survival, invasion, and angiogenesis, and contributes to local immune suppression. We show that systemic MFG-E8 blockade cooperates with cytotoxic chemotherapy, molecularly targeted therapy, and radiation therapy to induce destruction of various types of established mouse tumors. The combination treatments evoke extensive tumor cell apoptosis that is coupled to efficient dendritic cell cross-presentation of dying tumor cells. This linkage engenders potent antitumor effector T cells but inhibits FoxP3(+) T reg cells, thereby achieving long-term protective immunity. Collectively, these findings suggest that systemic MFG-E8 blockade might intensify the antitumor activities of existing therapeutic regimens through coordinated cell-autonomous and immune-mediated mechanisms.

Anti-amyloid activity of the C-terminal domain of proSP-C against amyloid beta-peptide and medin.

Amyloid fibrils are found in approximately 25 different diseases, including Alzheimer's disease. Lung surfactant protein C (SP-C) forms fibrils in association with pulmonary disease. It was recently found that the C-terminal domain of proSP-C (CTC), which is localized to the endoplasmic reticulum (ER) lumen, protects the transmembrane (TM) part of (pro)SP-C from aggregation into amyloid until it has a folded into an alpha-helix. CTC appears to have a more general anti-amyloid effect by also acting on TM regions of other proteins. Here we investigate interactions of CTC with the amyloid beta-peptide (Abeta) associated with Alzheimer's disease and medin, a peptide that forms fibrils in the most common form of human amyloid. CTC prevents fibril formation in Abeta and medin and forms a complex with Abeta oligomers, as judged by size-exclusion chromatography and electrospray ionization mass spectrometry. These data suggest that CTC functions as a chaperone that acts preferentially against unfolded TM segments and structural motifs found during amyloid fibril formation, a mechanism that may be exploited in forming a basis for future anti-amyloid therapy.

The source and secretion of immunoactive relaxin in rat milk.

The milk of many mammalian species contains hormones and growth factors in addition to nutrients and immunocompetent substances. These factors can be absorbed into the circulation of suckling neonates to exert important effects on metabolism and promote tissue and organ growth. Frequently, there is uncertainty as to whether such substances are gene products of the mammary glands themselves or are produced elsewhere and concentrated from the systemic circulation. The 6 kD polypeptide, relaxin, appears in milk of several mammalian species, including that of the rat, but proof of its source of secretion (corpus luteum vs. mammary gland) is so far lacking. The specific monoclonal anti-rat relaxin antibody MCA1 has previously been utilized successfully to investigate many of relaxin's actions in the rat, including those affecting the development of the mammary apparatus. In this report, MCA1 was utilized to aid in the identification of the source of relaxin in rat milk. Treatment of lactating rats with MCA1 completely neutralized the luteal relaxin circulating in serum but did not decrease the concentration of immunoactive relaxin secreted in milk. Moreover, the antibody did not appear to reach the mammary epithelium. The evidence thus supports the view that in the rat, the relaxin secreted in milk is primarily a product of the mammary glands and not concentrated from the systemic circulation.

Milk fat globule epidermal growth factor-factor VIII is down-regulated in sepsis via the lipopolysaccharide-CD14 pathway.

Phagocytosis prevents the release of potentially harmful or immunogenic materials from dying cells. Milk fat globule epidermal growth factor (EGF)-factor VIII (MFG-E8) mediates the clearance of apoptotic cells. We have previously shown that the administration of MFG-E8-rich exosomes from immature dendritic cells promotes the phagocytosis of apoptotic cells and improves survival in sepsis. Because endotoxin is elevated in polymicrobial sepsis, we hypothesized that down-regulation of MFG-E8 is mediated via the LPS-CD14 pathway, eventually leading to the accruement of apoptotic cells. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) in CD14-deficient (CD14(-/-)), TLR4-mutated and wild-type (WT) mice. In addition, endotoxemia was elicited by i.p. injection of LPS. LPS was also neutralized by pretreating CLP-induced WT mice with polymyxin B. Splenic MFG-E8 expression, phagocytic activity, and apoptosis were assessed 5 and 20 h after CLP or 5 h after LPS administration. In septic WT mice, MFG-E8 mRNA and protein levels were suppressed by 49 and 33%, respectively. Endotoxemia reduced MFG-E8 mRNA expression in a dose dependent manner and the down-regulation of MFG-E8 mRNA expression in CLP-induced sepsis was attenuated by polymyxin B. This CLP-induced suppression was not observed in both CD14(-/-) and TLR4-mutated mice. CLP significantly decreased phagocytic activity of peritoneal macrophages in WT (by 30%), but not in CD14(-/-) mice. CLP also induced significant apoptosis in the spleen of WT (by 61%), but less in CD14(-/-) mice. Thus, MFG-E8 production is down-regulated in sepsis by LPS-CD14 dependent fashion, leading to a reduction of phagocytosis of apoptotic cells.